Cytotoxic T-lymphocytes (CTL) are essential for immune control in HIV infection, and immunodominant CTL responses have been characterized for many HLA alleles. However the reasons for relative immunodominance of certain epitopes have not been defined, and the steps in antigen processing that shape an immunodominant epitope are not known. Moreover no studies have examined how HLA-restricted mutations that accumulate in HIV-1 infected people affect the generation of epitopes. Our long-term goal is to understand the rules that govern degradation and presentation of specific HIV antigens and the impairment of antigen presentation through mutations. In preliminary studies, we have identified allele-specific mutations in the flanking regions of dominant HIV epitopes that impair antigen processing and recognition by CTL. We have shown that an HLA-restricted mutation can affect trimming of longer peptide precursors in the endoplasmic reticulum (ER), thereby altering the relative availability of epitopes for class I loading. We hypothesize that antigen processing influences the hierarchy observed among epitopes and that specific HLA-associated mutations in flanking regions of epitopes alter distinct steps of the processing and serve as a mechanism for immune escape. This proposal will focus on a detailed analysis of changes in antigen processing pathways that may lead to viral escape. The specific aims include: 1. To define cytosolic processing ofCD8 T cell epitopes, focusing on HLA-A3/A11-restricted responses for initial studies; 2. To determine the extent to which N-glycosylation of peptides in the ER serves as a potential mechanism of viral escape from CTL responses. These goals are based on a common experimental design. We will follow the complete processing of HIV epitopes of interest in antigen presenting cells or reconstruct this process with subcellular fractions. We will analyze the degradation of HIV polypeptides in the cytosol by the proteasome and other peptidases, the translocation into the ER and the trimming and potential N-glycosylation of N-extended peptides in the ER lumen, the loading of peptides onto MHC-I molecules and their subsequent presentation to CTL. Given the extreme mutation rate of HIV and the persistence of immune selection pressure, these studies represent a critical first step in determining the role of antigen processing in immune escape, which will be important not only for the understanding of HIV immunopathogenesis but also for the design of candidate vaccines. [unreadable] [unreadable]